A-myb null mutant transgenic mice

ABSTRACT

Transgenic non-human animals and transgenic non-human stem cells are described having a functionally disrupted A-myb locus. Targeting constructs used to produce such transgenic stem cells and animals, and methods and targeting constructs for inactivating an endogenous A-myb gene locus, are also provided. Also provided are methods for generating transgenic sperm and transgenic nonhuman animals harboring a desired transgene.

This application is a 371 of PCT/US98/06896 filed Apr. 7, 1998, whichclaims the benefit of U.S. Provisional Application Serial No. 60/043,353filed Apr. 15, 1997.

FIELD OF THE INVENTION

The invention relates to transgenic non-human animals and transgenicnon-human animal cells harboring a transgene containing a mutation inthe A-myb gene and having a functionally disrupted A-myb gene locus. Theinvention further relates to transgenes and targeting constructs used toproduce such transgenic animals and cells, methods of using such animalsfor modeling male infertility disorders, and methods for using suchanimals to produce transgenic nonhuman animals and cells including afurther transgene.

BACKGROUND OF THE INVENTION

The myb gene family currently consists of three members, named A, B andc-myb. Of these, c-myb is the most extensively studied member. The B-myband A-myb genes share extensive sequence homology with c-myb.

The myb oncogene was first identified as the transforming gene of AvianMyeloblastosis virus (AMV) which causes myeloblastic leukemia inchickens and transforms myelomonocytic cells in culture (Baluda et al.,Virology 15: 185-199 (1964); C. Moscovici, Immunol. 71: 79-101 (1975)).The normal cellular counterpart of this oncogene, c-myb, is highlyconserved and is present in all vertebrate and some invertebrate speciesexamined (Franchini et al., Proc. Nat. Acad. Sci. USA 80: 7385-7389(1983); Katzen et al., Cell 41: 449-456 (1985)). Proteins encoded by theviral as well as the cellular myb gene appear to be localized in thenucleus, and these proteins exhibit a sequence-specific DNA-bindingactivity (Klempnauer et al., Cell 37: 537-547 (1984); Boyle et al.,Proc. Nat. Acad. Sci. USA 81: 42654269 (1984); Moelling et al., Cell 40:983-990 (1985); Biedenkapp et al., Nature 335: 835-837 (1988)). Theirsequence-specific DNA binding activity and ability to activatetranscription of reporter genes linked to certain promoter/enhancersequences suggest that they act as nuclear transcription factors (Sakuraet al., Proc. Nat. Acad. Sci. USA 86: 5758-5762 (1989); Dudek et al.,Proc. Nat. Acad. Sci. USA 89: 1291-1295 (1992)). A-myb in particular hasbeen recognized as a potent transactivator of transcription (Golay etal., Oncogene 9: 2469-2479 (1994); Foos et al., Oncogene 9: 2481-2488(1994)). Elimination of c-myb function in vivo, using gene-knock outtechniques, has indicated that homozygous c-myb mutant mice fail to showeffective fetal hepatic hematopoiesis resulting in the death of mice inutero confirming an essential role for c-myb in fetal hematopoiesis(Mucenski et al., Cell 65: 677-689 (1991)).

In contrast to c-myb, whose role in hematopoiesis is well established,little is known about the role of the A-myb gene in development. HumanA-myb is expressed in a variety of lymphoid and solid tumors (Shen-Onget al., Mol. Cell. Biol. 6: 380-392 (1986)). Foos et al., Oncogene 9:2481-2488 (1994) have reported ubiquitous expression of A-myb in chickencell lines. On the other hand, Sleeman, Oncogene 8: 1931-194 (1993)reported specific expression of Xenopus A-myb in testis, with very lowlevels of expression in ovarian tissue.

Murine spermatogenesis is divided into three distinct intervals whichinclude: (1) stem cell proliferation and renewal; (2) meiosis and (3)germ cell differentiation (spermatogenesis). Spermatogenesis in miceoccurs in the seminiferous tubule, a specialized epithelium in whichspermatogonia are located in close proximity to the basement membrane.Cells at progressively later stages of meiosis and differentiation aresituated closer to the tubular lumen. Spermatogenesis in the mouseoccurs in twelve distinct histological stages. Each stage consists of aconstant pattern of germ cell association. Stage VII of mousespermatogenesis is a testosterone dependent stage and includes thefollowing cell types: Type A spermatogonia (stem cells) along withpreleptotene spermatocytes, usually situated closest to the basementmembrane; pachytene spermatocytes (early meiotic cells) located at theintermediate position between basement membrane and the lumen; and step7 spermatids and step 16 spermatozoa located closest to the lumen.

Recently, it has been demonstrated that A-myb is expressed at highlevels in mouse testis where it is transcribed as multiple transcripts,some of which are differentially spliced to code for smaller proteins(Mettus et al., Oncogene 9: 3077-3086 (1994)). A high level ofexpression of A-myb was seen in mouse testis and very low levels ofexpression were detected in mouse spleen, ovary and brain. Asdifferentiation proceeds and the primary spermatogonia mature intosecondary spermatogonia, which in turn maturate into spermatocytes, adistinct downregulation of A-myb expression was seen in in Situhybridization studies. A-myb was maximally expressed in type Aspermatogonia which are located proximal to the basement membrane andpreleptotene and pachytene spermatocytes located between the basementmembrane and the lumen. Less intense hybridization was also seen withspermatids. Thus, A-myb expression was maximal in proliferating stemcells and early meiotic cells but reduced in spermatids and absent inspermatozoa undergoing terminal differentiation.

Despite these findings, the functional significance of A-myb remains tobe established, particularly in spermatogenesis. More completeinformation concerning the function of A-myb requires studying theeffect of the encoded protein, or the lack thereof, in vivo.

Various animals have been produced with germ line foreign DNA, or withaltered levels of expression of certain genes. These animals typicallyhave a foreign or mutated gene incorporated into their genome. In onesuch class of transgenic animal, the so-called homozygous null or“knockout” mutants, expression of an endogenous gene has been suppressedthrough genetic manipulation.

Transgenic animals generally harbor at least one copy of a transgeneeither homologously or nonhomologously integrated into an endogenouschromosomal location so as to encode a foreign or mutant protein. Suchtransgenic animals are usually produced by introducing the transgene ortargeting construct into a fertilized egg, or into an embryonic stem(ES) cell which is then injected into an embryo. Introduction of thetransgene into the fertilized egg or ES cell is typically performed bymicroinjection, retroviral infection, electroporation, lipofection, orbiolistics. The fertilized egg or embryo is then transferred to anappropriate pseudopregnant female for the duration of gestation.Knockout mutants may be obtained according to this method where thenon-native DNA which is introduced comprises a nucleic acid constructthat will be used to suppress expression of a particular gene. Suchknockout constructs are typically introduced into ES cells.

One problem in the production of transgenic animals is the relativelylow rate of success in obtaining incorporation of the transgene into thegermline of the host species. Moreover, while transgenes have beenincorporated into fertilized eggs by microinjection, the smaller size ofsperm cells makes incorporation of transgenes by injection difficult.What is needed is a method to increase the frequency of first generationtransgenic offspring and to provide for the incorporation of transgenesinto sperm.

Male infertility continues to be significant reproductive healthproblem. What is needed is a live animal model which may be used for thestudy of male infertility, and for screening and evaluation of potentialtherapeutic agents useful in the treatment of this disorder.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide nonhumananimals in which expression of the A-myb gene has been suppressed.

It is an object of the invention to provide nonhuman cells and nonhumananimals containing a homozygous null mutation of the A-myb gene locus.

It is an object of the invention to provide constructs and vectors forproducing such cells and animals containing an A-myb homozygous nullmutation.

It is a further object of the invention to provide a method forobtaining incorporation of transgenes of interest into sperm cells, andto provide sperm cells so transformed.

It is an object of the invention to provide a method for the productionof nonhuman transgenic animals, by utilizing the aforesaid transgenicsperm.

These and other objects of the invention will be apparent to those ofordinary skill in the art from the following disclosure.

In accordance with the foregoing objects, the invention in one aspect isa targeting construct for functionally disrupting an A-myb gene. Thetargeting construct comprises a polynucleotide containing at least oneportion having a sequence that is substantially homologous to a sequencepresent in or flanking an A-myb gene locus and which, when integrated atthe corresponding A-myb gene locus, functionally disrupts expression ofA-myb protein from the gene locus. Such targeting constructs, orportions thereof, integrate at the A-myb gene locus by homologousrecombination between the endogenous gene locus and the targetingconstruct.

In one embodiment, the A-myb gene is functionally disrupted by atargeting construct which inserts a sequence, typically into a codingsequence (i.e., exon), wherein the resultant disrupted A-myb gene issubstantially incapable of expressing a functional A-myb protein. In onesuch embodiment, the targeting construct comprises an upstream homologyregion having a sequence with substantial identity to a first endogenousA-myb gene sequence, a nonhomologous replacement portion, a downstreamhomology region having a sequence with substantial identity to a secondendogenous A-myb gene sequence located downstream from said firstendogenous A-myb sequence, wherein the upstream homology region anddownstream homology region flank the nonhomologous replacement portion.

The nonhomologous replacement portion of the targeting constructadvantageously comprises a positive selection expression cassette, suchas neo. The targeting construct further advantageously comprises anegative selection cassette distal to either the upstream homologyregion or the downstream homology region. The negative selectioncassette may comprise, for example, a tk gene.

According to another embodiment, the invention provides a method forgenerating stem cells having a functionally disrupted endogenous A-mybgene comprising transferring the aforesaid targeting construct intopluripotent stem cells, and selecting for stem cells having a correctlytargeted homologous recombination between the targeting construct and anendogenous A-myb gene sequence.

According to yet another embodiment, the invention provides a method forgenerating nonhuman animals having a functionally disrupted endogenousA-myb gene, comprising the steps of transferring, into a nonhumanblastocyst, stem cells having a correctly targeted homologousrecombination between the aforesaid targeting construct and anendogenous A-myb gene sequence; implanting the resultant blastocyst intoa pseudopregnant female; and collecting offspring harboring anendogenous A-myb allele having the correctly targeted homologousrecombination.

According to-another embodiment, the invention provides transgenicnonhuman animals and stem cells having a genome comprising at least onefunctionally disrupted A-myb gene. The animal or stem cell is preferablyhomozygous for the functionally disrupted A-myb gene. Such a homozygoustransgenic animal or stem cell is substantially incapable of directingthe efficient expression of endogenous A-myb. For example, in apreferred embodiment, a transgenic mouse is homozygous for aninactivated endogenous (i.e., naturally occurring) A-myb gene.

According to one embodiment, the transgenic nonhuman animal or stem cellhomozygous for a functionally disrupted A-myb gene comprise an A-mybgene disrupted by an integrated targeting construct, e.g., an integratedtargeting construct comprising a neo gene.

According to a preferred embodiment of the invention, the transgenicanimal is a mouse comprising a genome having a functionally disruptedmurine A-myb allele. Preferably, the mouse is homozygous for thefunctionally disrupted A-myb allele. Such mice do not produce functionalA-myb protein and are infertile.

According to another embodiment, a method is provided for generatingnonhuman animals producing sperm harboring a desired transgene.

Spermatogonia are obtained from a nonhuman animal which is homozygousfor a functionally disrupted endogenous A-myb gene. An A-myb constructcomprising a first DNA sequence encoding a functional A-Myb polypeptideand a second DNA sequence encoding the desired transgene of interest, istransferred into the spermatogonia. The spermatogonia harboring theA-myb construct are then introduced into the testes of nonhuman animalswhich are homozygous for a functionally disrupted endogenous A-myb gene.Fertile individuals are then selected from the animals having receivedthe transfected spermatogonia. Fertile individuals produce spermharboring the desired transgene.

According to another method for generating nonhuman animals producingsperm harboring a desired transgene, the testis of nonhuman animalswhich are homozygous for a functionally disrupted endogenous A-myb geneare infected with an expression vector directing the incorporation intothe DNA of said infected testes a first DNA sequence encoding afunctional A-Myb polypeptide and a second DNA sequence encoding thedesired transgene of interest, linked to said first DNA sequence.Fertile individuals are selected from the infected animals. The fertileindividuals produce sperm harboring the desired transgene.

The present invention also provides for the treatment of maleinfertility in those occurrences of the disease which arise from adefect in the A-myb locus. Treatment comprises the transfer of DNAencoding functional A-Myb polypeptide to the cells of the testes, or byadministration of functional A-Myb polypeptide directly to the testes.

According to one such treatment method for restoring fertility in asubject who is infertile due to a defect in the A-myb locus,spermatogonia is first obtained from the subject. An A-myb construct istransferred into the obtained spermatogonia. The A-myb constructcomprises a DNA sequence encoding a functional A-Myb polypeptide. Thespermatogonia harboring the A-myb construct encoding the functionalA-Myb polypeptide is introduced into the testes of the individual toobtain production of functional A-Myb polypeptide in the testes.

In another embodiment of an infertility treatment method, fertility in asubject who is infertile due to a defect in the A-myb locus is restoredby infecting the testis of the individual with a retrovirus vector. Thevector directs the incorporation of a DNA sequence encoding a functionalA-Myb polypeptide into the DNA of the infected testes.

In yet another embodiment, fertility is restored in a subject who isinfertile due to a defect in the A-myb locus by locally administered afunctional A-Myb polypeptide to the testes of the subject, such as byinjection into the seminiferous tubules.

The invention is also directed to spermatogonia comprising recombinantDNA encoding a functional A-Myb polypeptide.

As used herein, the term “A-myb gene” or “A-myb gene locus” refers to aregion of a chromosome spanning all of the exons which potentiallyencode the A-myb polypeptide and extending through flanking sequences(e.g., including promoters, enhancers, etc.) that participate in A-mybprotein expression. Thus, an A-myb gene locus includes the regionspanning from the first exon through the last exon and also includesadjacent flanking sequences (e.g., polyadenylation signals) that mayparticipate in A-myb gene expression.

The terms “functional disruption” or “functionally disrupted” as usedherein means that a gene locus comprises at least one mutation orstructural alteration such that the functionally disrupted gene issubstantially incapable of directing the efficient expression offunctional gene product. By way of example but not limitation, anendogenous A-myb gene that has a neo gene cassette integrated into anexon of an A-myb gene is not capable of encoding a functional A-mybprotein and is therefore a functionally disrupted A-myb gene locus.Deletion or interruption of essential transcriptional regulatoryelements, polyadenylation signals(s), splicing site sequences will alsoyield a functionally disrupted gene. Functional disruption of anendogenous A-myb gene, may also be produced by other methods (e.g.,antisense polynucleotide gene suppression). The term “structurallydisrupted” refers to a targeted gene wherein at least one structural(i.e., exon) sequence has been altered by homologous gene targeting(e.g., by insertion, deletion, point mutation(s), and/or rearrangement).Typically, alleles that are structurally disrupted are consequentlyfunctionally disrupted. However A-myb alleles may also be functionallydisrupted without concomitantly being structurally disrupted, i.e., bytargeted alteration of a non-exon sequence such as ablation of apromoter. An allele comprising a targeted alteration that interfereswith the efficient expression of a functional gene product from theallele is referred to as a “null allele”.

The expression “functional A-Myb polypeptide” means a polypeptide which,upon expression in or administration to A-myb^(−/−) male individuals, issufficient to restore spermatogenesis and fertility in such individuals.

The term “corresponds to” is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence.

The term “complementary to” is used herein to mean that the subjectsequence is homologous to all or a portion of a reference polynucleotidesequence. For illustration, the nucleotide sequence “TATAC” correspondsto a reference sequence “TATAC” and is complementary to a referencesequence “GTATA”.

The terms “substantially corresponds to”, “substantially homologous”, or“substantial identity” as used herein denotes a characteristic of anucleic acid sequence, wherein a nucleic acid sequence has at leastabout 70 percent sequence identity as compared to a reference sequence,typically at least about 85 percent sequence identity, and preferably atleast about 95 percent sequence identity as compared to a referencesequence. The percentage of sequence identity is calculated excludingsmall deletions or additions which total less than 25 percent of thereference sequence. The reference sequence may be a subset of a largersequence, such as a portion of a gene or flanking sequence, or arepetitive portion of a chromosome. However, the reference sequence isat least 18 nucleotides long, typically at least about 30 nucleotideslong, and preferably at least about 50 to 100 nucleotides long.

“Substantially complementary” as used herein refers to a sequence thatis complementary to a sequence that substantially corresponds to areference sequence. In general, targeting efficiency increases with thelength of the targeting transgene portion (i.e., homology region) thatis substantially complementary to a reference sequence present in thetarget DNA (i.e., crossover target sequence). In general, targetingefficiency is optimized with the use of isogeneic DNA homology clamps,although it is recognized that the presence of various recombinases mayreduce the degree of sequence identity required for efficientrecombination.

The term “nonhomologous sequence”, as used herein, has both a generaland a specific meaning, it refers generally to a sequence that is notsubstantially identical to a specified reference sequence, and where noparticular reference sequence is explicitly identified, it refersspecifically to a sequence that is not substantially identical to asequence of at least about 50 contiguous bases at an endogenous A-mybgene.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring. As used herein, laboratory strains of rodents whichmay have been selectively bred according to classical genetics areconsidered naturally-occurring animals.

As used herein, the term “targeting construct” refers to apolynucleotide which comprises: (1) at least one homology region havinga sequence that is substantially identical to or substantiallycomplementary to a sequence present in a host cell A-myb gene locus, and(2) a targeting region which becomes integrated into an host cell A-mybgene locus by homologous recombination between a targeting constructhomology region and said A-myb gene locus sequence. If the targetingconstruct is a “hit-and-run” or “in-and-out” type construct (Valanciusand Smithies (1991) Mol. Cell. Biol. 11: 1402; Donehower et al. (1992)Nature 356: 215; (1991) J. NIH Res. 3: 59; which are incorporated hereinby reference), the targeting region is only transiently incorporatedinto the endogenous A-myb gene locus and is eliminated from the hostgenome by selection. A targeting region may comprise a sequence that issubstantially homologous to the endogenous A-myb gene sequence and/ormay comprise a nonhomologous sequence, such as a selectable marker(i.e., neo, tk, gkt). The term “targeting construct” does notnecessarily indicate that the polynucleotide comprises a gene whichbecomes integrated into the host genome, nor does it necessarilyindicate that the polynucleotide comprises a complete structural genesequence. As used in the art, the term “targeting construct” issynonymous with the term “targeting transgene”.

The terms “homology region” and “homology clamp” as used herein refer toa segment (i.e., a portion) of a targeting construct having a sequencethat substantially corresponds to, or is substantially complementary to,a predetermined A-myb gene sequence, which can include sequencesflanking said A-myb. A homology region is generally at least about 100nucleotides long, preferably at least about 250 to 500 nucleotides long,typically at least about 1000 nucleotides long or longer.

The terms “crossover target sequences” or “endogenous target sequences”as used herein refer to A-myb gene sequences that substantiallycorrespond to, or are substantially complementary to, a transgenehomology region.

As used herein, the term “targeting region” refers to a portion of atargeting construct which becomes integrated into an endogenouschromosomal location following homologous recombination between ahomology clamp and an endogenous A-myb gene sequence. Typically, atargeting region is flanked on each side by a homology clamp, such thata double-crossover recombination between each of the homology clamps andtheir corresponding endogenous A-myb gene sequences results inreplacement of the portion of the endogenous A-myb gene locus by thetargeting region; in such double-crossover gene replacement targetingconstructs the targeting region can be referred to as a “replacementregion”. However, some targeting constructs may employ only a singlehomology clamp (e.g., some “hit-and-run”-type vectors, see, Bradley etal. (1992) Bio/Technology 10: 534, incorporated herein by reference).

As used herein, the term “replacement region” refers to a portion of atargeting construct flanked by homology regions. Upon double-crossoverhomologous recombination between flanking homology regions and theircorresponding endogenous A-myb gene crossover target sequences, thereplacement region is integrated into the host cell chromosome betweenthe endogenous crossover target sequences. Replacement regions can behomologous (e.g., have a sequence similar to the endogenous A-myb genesequence but having a point mutation or missense mutation),nonhomologous (e.g., a neo gene expression cassette), or a combinationof homologous and nonhomologous regions.

DESCRIPTION OF THE FIGURES

FIG. 1 is a restriction map of an A-myb genomic clone (top), and thestructure of a targeting vector (bottom). The coding exons are depictedby black boxes. The black boxes in the restriction map represent A-mybexons 3, 4 and 5. A genomic fragment comprising the entire exon 3 and aportion of intron 3 was obtained by digestion of the clone with SspI.The clone, which was used as a probe, is shown as a black bar. Thearrows depict the transcriptional orientation of the neo (neomycintransferase gene) and tk (thymidine kinase) genes. Bg, BgIII; S, SspI;Ev, EcoRV; E, EcoRI; C, ClaI; N, NcoI; Ba, BanI; Xb, XbaI; St, StuI; Bs,BstXII; H, HindIII; B, BamHI.

FIG. 2 is a Southern blot analysis of genomic DNA extracts from ES cellclones electroporated with the targeting vector of FIG. 1. The DNA wasdigested with HindIII, fractionated on an agarose gel, blotted onto anitrocellulose paper and hybridized with the ³²P-labeled SspI fragmentindicated as a black bar in FIG. 1. Molecular weight markers (γDNAdigested with HindIII) are shown on the left. Lanes 4 and 5 contain DNAfrom ES cell clones that contain a disrupted A-myb locus.

FIG. 3 is a Southern blot analysis of the DNA extracted from tailbiopsies of 10-day old pups of A-myb^(+/−) intercrosses, digested withHindIII, and blotted onto a nitrocellulose membrane. The blots werehybridized with the same ³²P-labeled probe as in the previous figure.The A-myb genotypes of the animals are presented above the lanes.A-myb^(+/+) (6 kb); A-myb^(+/−) (6 kb+8 kb); A-myb^(−/−) (8 kb).

FIG. 4 is a Western blot analysis of extracts prepared from A-myb^(+/+),A-myb^(+/−) and A-myb^(−/−) mouse testes. The blot was probed with arabbit polyclonal anti-Myb antibody developed by enhancedchemiluminescence. The genotypes of the mouse testes from which thetestes extracts were prepared are shown above the lanes.

FIG. 5A is a comparison of the total body size and testicular size of 4week-old A-myb^(+/−) (left) and A-myb^(−/−) (right) mice from the samelitter. The testes dissected from the same mice are shown below themice.

FIG. 5B is a comparison of the total body size and testicular size of 10week-old A-myb^(+/+) (left), A-myb^(+/−) (middle) and A-myb^(−/−)(right) mice from the same litter. The testes dissected from the samemice are shown below the mice.

FIG. 6A is a 100× view of a hematoxylin and eosin-stained section of theseminiferous tubules of A-myb^(+/+) mouse testis.

FIG. 6B is a 100× view of a hematoxylin and eosin-stained section of theseminiferous tubules of A-myb^(−/−) mouse testis.

FIG. 7A is a 25× magnification cross-section of wild-type mouse ovaries.

FIG. 7B is a 25× magnification cross-section of A-myb^(−/−) mouseovaries.

FIG. 8A is a 10× magnification cross-section of A-myb^(+/−) female mouseepithelium, taken from breast tissue of maternal mice two days followingthe delivery of pups.

FIG. 8B is a 20× view of the same A-myb^(+/−) tissue of FIG. 8A.

FIG. 8C is a 10× magnification cross-section of A-myb^(−/−) female mouseepithelium, taken from breast tissue of maternal mice two days followingthe delivery of pups.

FIG. 8D is a 20× magnification view of the same A-myb^(−/−) tissue ofFIG. 8C.

FIG. 9A shows the structure of the pMV-7 vector of Kirschmeier et al.,DNA 7, 219-225 (1988).

FIG. 9B shows the structure of the modified pMV-7 vector pMV-7 ΔClaI/neo E/H, generated by excision of the neo cassette from pMV-7 ofFIG. 9A by ClaI digest and placement of the cassette at the Eco RI/HindIII cloning site of pMV-7.

FIG. 9C shows the structure of the vector pMV-7 Δ ClaI/neo E/H-A-myb,formed by insertion of the 6.7 kbp fragment SEQ ID NO:4 into the ClaIsite of the FIG. 9B vector. The insert contains the A-myb promoter(cross-hatch), the A-myb coding sequence, and a polyadenylation signalfrom the bovine growth hormone gene (solid). Arrows in FIGS. 9A-9Cindicate transcription start sites.

DETAILED DESCRIPTION OF THE INVENTION

Chimeric targeted mice may be derived according to Hogan, et al.,Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring HarborLaboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: APractical Approach, E. J. Robertson, ed., IRL Press, Washington, D.C.,(1987) which are incorporated herein by reference.

Embryonic stem cells may be manipulated according to publishedprocedures (Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed., IRL Press, Washington, D.C. (1987);Zjilstra et al., Nature 342:435-438 (1989); and Schwartzberg et al.,Science 246:799-803 (1989), each of which is incorporated herein byreference).

Oligonucleotides can be synthesized on an Applied BioSystemsoligonucleotide synthesizer according to specifications provided by themanufacturer.

According to the practice of the invention, the endogenous A-myb allelesof a cell line or nonhuman animal are functionally disrupted so thatexpression of endogenously encoded A-myb gene is suppressed oreliminated. In general, polynucleotide constructs are employed for thispurpose. Methods for accomplishing this result are described in detailin WO 95/11968 with respect to transgenic non-human animals andmammalian cells hosting a transgene encoding an amyloid precursorprotein (APP) and “knock out” mutants thereof. The entire disclosure ofWO 95/11968 is incorporated herein by reference. Similar methods ofpreparing transgenic non-human animals and mammalian cells characterizedby knock-out mutations are described in WO 94/06908 (targeting oflymphocyte transduction gene expression) and, WO 94/28123 (targeting ofCD28 expression), the entire disclosures of which are incorporatedherein by reference.

Gene targeting, which is a method of using homologous recombination tomodify a mammalian genome, can be used to introduce genetic changes intocultured cells. By targeting a gene of interest in embryonic stem (ES)cells, these changes can be introduced into the germlines of laboratoryanimals to study the effects of the modifications on whole organisms,among other uses. The gene targeting procedure is accomplished byintroducing into tissue culture cells a DNA targeting construct that hasa segment homologous to a target locus and which also comprises anintended sequence modification (e.g., insertion, deletion, pointmutation). The treated cells are then screened for accurate targeting toidentify and isolate those which have been properly targeted. A commonscheme to disrupt gene function by gene targeting in ES cells is toconstruct a targeting construct which is designed to undergo ahomologous recombination with its chromosomal counterpart in the ES cellgenome. The targeting constructs are typically arranged so that theyinsert an additional sequence, such as a positive selection marker, intocoding elements of the target gene, thereby functionally disrupting it.Targeting constructs usually are insertion-type or replacement-typeconstructs (Hasty et al. (1991) Mol. Cell. Biol. 11: 4509).

The invention encompasses production of stem cells and nonhuman animalsthat have the endogenous A-myb gene inactivated by gene targeting with ahomologous recombination targeting construct. The A-myb gene sequencemay be used as a basis for producing PCR primers that flank a regionthat will be used as a homology clamp in a targeting construct. The PCRprimers are then used to amplify a genomic sequence from a genomic clonelibrary or from a preparation of genomic DNA, preferably from the strainof nonhuman animal that is to be targeted with the targeting construct.The amplified DNA is then used as a homology clamp and/or targetingregion. General principles regarding the construction of targetingconstructs and selection methods are reviewed in Bradley et al. (1992)Bio/Technology 10: 534, incorporated herein by reference.

The isolation of A-myb genomic DNA useful for this purpose is describedherein. Appropriate probes may be designed based on known A-myb cDNAnucleotide sequences. For example, the complete nucleotide sequence ofthe mouse A-myb cDNA (SEQ ID NO:1), deduced amino acid sequence (SEQ IDNO:2), and cDNA restriction map are disclosed in Mettus et al., Oncogene9: 3077-3086 (1994), the entire disclosure of which is incorporatedherein by reference. The encoded mouse A-myb protein contains 751 aminoacids (SEQ ID NO:2) and has an estimated molecular weight of 83 kDa. Itmay be appreciated that A-myb genomic DNA may be derived using anappropriate cDNA fragment as a probe to identify and isolate genomicA-myb from an appropriate genomic DNA library.

Targeting constructs can be transferred into pluripotent stem cells,such as ES cells, wherein the targeting constructs homologouslyrecombine with a portion of the endogenous A-myb gene locus and createmutation(s) (i.e., insertions, deletions, rearrangements, sequencereplacements, and/or point mutations) which prevent the functionalexpression of the endogenous A-myb gene.

One method is to delete, by targeted homologous recombination, essentialstructural elements of the endogenous A-myb gene. For example, atargeting construct can homologously recombine with an endogenous A-mybgene and delete a portion spanning substantially all of one or moreexons to create an exon-depleted allele, typically by inserting areplacement region lacking the corresponding exon(s). Transgenic animalshomozygous for the exon-depleted allele (e.g., by breeding ofheterozygotes to each other) are essentially incapable of expressing afunctional endogenous A-myb polypeptide. Similarly, homologous genetargeting can be used, if desired, to functionally disrupt the A-mybgene by deleting only a portion of an exon.

Targeting constructs can also be used to delete essential regulatoryelements of the endogenous A-myb gene, such as promoters, enhancers,splice sites, polyadenylation sites, and other regulatory sequences,including cis-acting sequences that may occur upstream or downstream ofthe A-myb structural gene but which participate in endogenous A-myb geneexpression. Deletion of regulatory elements is typically accomplished byinserting, by homologous double-crossover recombination, a replacementregion lacking the corresponding regulatory element(s).

The mouse A-myb gene was isolated by screening a λ DASH mouse genomiclibrary derived from the 129/J mouse strain, using a probe derived fromthe 5′ end of the A-myb cDNA clone (Mettus et al., Oncogene 9,3077-3086, 1994) that encodes the DNA binding domain of the protein.Positive clones that contained a 5.9 kbp HindIII fragment were subclonedinto pGEM 7Zf(+) plasmid vector. The complete nucleotide sequence ofthis 5.9 kbp clone was determined using the method of Sanger et al.,Proc. Natl. Acad. Sci. USA 74, 5463-5467 (1977). The sequence analysisshowed that this fragment contained exons 3,4 and 5 of the gene thatcode for the 5′ end of the DNA binding domain of the protein (FIG. 1,top). The complete nucleotide sequence of this 5.9 kbp clone is SEQ IDNO:3.

While the A-myb promoter does not contain a putative TATA or CCAT box,it contains a highly GC rich sequence, a feature observed with manytestis-specific and housekeeping gene promoters.

A preferred method is to interrupt essential structural and/orregulatory elements of the endogenous A-myb gene by targeted insertionof a polynucleotide sequence, and thereby functionally disrupt theendogenous A-myb gene. For example, a targeting construct canhomologously recombine with the endogenous A-myb gene and insert anonhomologous sequence, such as a neo expression cassettes into astructural element (e.g., an exon) and/or regulatory element (e.g.,enhancer, promoter, splice site, polyadenylation site) to yield atargeted A-myb allele having an insertional interruption. The insertedsequence can range in size from about 1 nucleotide (e.g., to produce aframeshift in an exon sequence) to several kilobases or more, as limitedby efficiency of homologous gene targeting with targeting constructshaving a long nonhomologous replacement region.

One preferred target site is the DNA binding domain of the A-myb gene.The DNA binding domain in the A-myb protein spans amino acidstryptophan-32 to valine-188, corresponding to nucleotides 352 to 820 ofthe murine A-myb cDNA. See SEQ ID NO:1.

Targeting constructs can also be employed to replace a portion of theendogenous A-myb gene with an exogenous sequence (i.e., a portion of atargeting transgene); for example, a first exon of an A-myb gene may bereplaced with a substantially identical portion that contains a nonsenseor missense mutation.

A targeting construct may be transferred by electroporation ofmicroinjection into a totipotent ES cell line. The targeting constructhomologously recombines with endogenous sequences in or flanking of theA-myb gene locus and functionally disrupts at least one allele of theA-myb gene. Typically, homologous recombination of the targetingconstruct with endogenous A-myb locus sequences will result inintegration of a nonhomologous sequence encoding and expressing aselectable marker, such as neo, usually in the form of a positiveselection cassette. ES cells having at least one such A-myb null alleleare selected for by propagating the cells in a medium that permits thepreferential propagation of cells expressing the selectable marker.Selected ES cells are examined by PCR analysis and/or Southern blotanalysis to verify the presence of a correctly targeted A-myb allele.Breeding of nonhuman animals which are heterozygous for a null allelemay be performed to produce nonhuman animals homozygous for said nullallele, so-called “knockout” animals (Donehower et al. (1992) Nature256: 215; Science 256: 1392, incorporated herein by reference).Alternatively ES cells homozygous for a null allele having an integratedselectable marker can be produced in culture by selection in a mediumcontaining high levels of the selection agent (e.g., G418 orhygromycin). Heterozygosity and/or homozygosity for a correctly targetednull allele can be verified with PCR analysis and/or Southern blotanalysis of DNA isolated from an aliquot of a selected ES cell cloneand/or from tail biopsies.

Gene targeting techniques which have been described, include but are notlimited to: co-electroporation, “hit-and-run”, single-crossoverintegration, and double-crossover recombination (Bradley et al. (1992)Bio/Technology 10: 534). The preparation of the homozygous A-myb nullmutants can be practiced using essentially any applicable homologousgene targeting strategy known in the art. The configuration of atargeting construct depends upon the specific targeting techniquechosen. For example, a targeting construct for single-crossoverintegration or “hit-and-run” targeting need only have a single homologyclamp linked to the targeting region, whereas a double-crossoverreplacement-type targeting construct requires two homology clamps, oneflanking each side of the replacement region.

For example and not limitation, a targeting construct comprises, inorder: (1) a first homology clamp having a sequence substantiallyidentical to a sequence within about 3 kilobases upstream (i.e., in thedirection opposite to the transnational reading frame of the exons) ofan exon of an endogenous A-myb gene, (2) a replacement region comprisinga positive selection cassette having a pgk promoter drivingtranscription of a neo gene, (3) a second homology clamp having asequence substantially identical to a sequence within about 3 kilobasesdownstream of said exon of said endogenous A-myb gene, and (4) anegative selection cassette, comprising a PGK promoter drivingtranscription of an HSV tk gene. Such a targeting construct is suitablefor double-crossover replacement recombination which deletes a portionof the endogenous A-myb locus spanning said exon and replaces it withthe replacement region having the positive selection cassette. Thedeleted exon is one which is essential for expression of a functionalA-myb gene product. Thus, the resultant exon-depleted allele isfunctionally disrupted and is termed a null allele.

Targeting constructs comprise at least one homology clamp linked inpolynucleotide linkage (i.e., by phosphodiester bonds) to a targetingregion. A homology clamp has a sequence which substantially correspondsto, or is substantially complementary to, an endogenous A-myb genesequence of a nonhuman host animal, and may comprise sequences flankingthe A-myb gene.

Although no lower or upper size boundaries for recombinogenic homologyclamps for gene targeting have been conclusively determined in the art,the best mode for homology clamps is believed to be in the range betweenabout 50 bp and several tens of kilobases. Consequently, targetingconstructs are generally at least about 50 to 100 nucleotides long,preferably at least about 250 to 500 nucleotides long, more preferablyat least abut 1000 to 2000 nucleotides long, or longer. Constructhomology regions (homology clamps) are generally at least about 50 to100 bases long, preferably at least about 100 to 500 bases long, andmore preferably at least about 750 to 2000 bases long. It is believedthat homology regions of about 7 to 8 kilobases in length are preferredwith one preferred embodiment having a first homology region of about 7kilobases flanking one side of a replacement region and a secondhomology region of abut 1 kilobase flanking the other side of saidreplacement region. The length of homology (i.e., substantial identity)for a homology region may be selected at the discretion of thepractitioner on the basis of the sequence composition and complexity ofthe endogenous A-myb gene target sequence(s) and guidance provided inthe art. Targeting constructs have at least one homology region having asequence that substantially corresponds to, or is substantiallycomplementary to, an endogenous A-myb gene sequence (e.g., an exonsequence, an enhancer, a promoter, an intronic sequence, or a flankingsequence within about 3-20 kb of the A-myb gene). Such a targetingconstruct homology region serves as a template for homologous pairingand recombination with substantially identical endogenous A-myb genesequence(s). In targeting constructs, such homology regions typicallyflank the replacement region, which is a region of the targetingconstruct that is to undergo replacement with the targeted endogenousA-myb gene sequence. Thus, a segment of the targeting construct flankedby homology regions can replace a segment of an endogenous A-myb genesequence by double-crossover homologous recombination. Homology regionsand targeting regions are linked together in conventional linearpolynucleotide linkage (5′ to 3′ phosphodiester backbone). Targetingconstructs are generally double-stranded DNA molecules, most usuallylinear.

Homology regions are generally used in the same orientation (i.e., theupstream direction is the same for each homology region of a transgeneto avoid rearrangements). Double-crossover replacement recombinationthus can be used to delete a portion of the endogenous A-myb andconcomitantly transfer a nonhomologous portion (i.e., a neo geneexpression cassette) into the corresponding chromosomal location.Double-crossover recombination can also be used to add a nonhomologousportion into the endogenous A-myb gene without deleting endogenouschromosomal portions. However, double-crossover recombination can alsobe employed simply to delete a portion of an endogenous gene sequencewithout transferring a nonhomologous portion into the endogenous A-mybgene. Upstream and/or downstream from the nonhomologous portion may be agene which provides for identification of whether a double-crossoverhomologous recombination has occurred; such a gene is typically the HSVtk gene which may be used for negative selection.

Typically, targeting constructs used for functionally disruptingendogenous A-myb genes will comprise at least two homology regionsseparated by a nonhomologous sequence which contains an expressioncassette encoding a selectable marker, such as neo (Smith and Berg(1984) Cold Spring Harbor Symp. Quant. Biol. 49: 171; Sedivy and Sharp(1989) Proc. Natl. Acad. Sci. (U.S.A.) 86: 227; Thomas and Capechi(1987), Cell 51: 503). However, some targeting transgenes may have thehomology region(s) flanking only one side of a nonhomologous sequence.Targeting transgenes of the invention may also be of the type referredto in the art as “hit-and-run” or “in-and-out” transgenes (Valancius andSmithies (1991) Mol. Cell. Biol. 11: 1402; Donehower et al. (1992)Nature 356: 215; (1991) J.NIH Res. 3: 59; which are incorporated hereinby reference).

The positive selection expression cassette encodes a selectable markerwhich affords a means for selecting cells which have integratedtargeting transgene sequences spanning the positive selection expressioncassette. The negative selection expression cassette encodes aselectable marker which affords a means for selecting cells which do nothave an integrated copy of the negative selection expression cassette.Thus, by a combination positive-negative selection protocol, it ispossible to select cells that have undergone homologous replacementrecombination and incorporated the portion of the transgene between thehomology regions (i.e., the replacement region) into a chromosomallocation by selecting for the presence of the positive marker and forthe absence of the negative marker (Valancius and Smithies, supra).

An expression cassette typically comprises a promoter which isoperational in the targeted host cell (e.g., ES cell) linked to astructural sequence that encodes a protein or polypeptide that confers aselectable phenotype on the targeted host cell, and a polyadenylationsignal. A promoter included in an expression cassette may beconstitutive, cell type-specific, stage-specific, and/or modulatable(e., by hormones such as glucocorticoids; MMTV promoter), but isexpressed prior to and/or during selection. An expression cassette canoptionally include one or more enhancers, typically linked upstream ofthe promoter and within about 3-10 kilobases. However, when homologousrecombination at the targeted endogenous site(s) places thenonhomologous sequence downstream of a functional endogenous promoter,it may be possible for the targeting construct replacement region tocomprise only a structural sequence encoding the selectable marker, andrely upon the endogenous promoter to drive transcription (Doetschman etal. (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 8583; incorporated hereinby reference). Similarly, an endogenous enhancer located near thetargeted endogenous site may be relied on to enhance transcription oftransgene sequences in enhancerless transgene constructs.

Preferred expression cassettes for inclusion in the targeting constructsencode and express a selectable drug resistance marker and/or a HSVthymidine kinase (tk) enzyme. Suitable drug resistance genes include,for example: gpt (xanthine-guanine phosphoribosytltransferase), whichcan be selected for with mycophenolic acid; neo (neomycinphosphotransferase), which can be selected for with G418 or hygromycin;and DFHR (dihydrofolate reductase), which can be selected for withmethotrexate (Mulligan and Berg (1981) Proc. Natl. Acad. Sci. (U.S.A.)78: 2072; Southern and Berg (1982) J. Mol. Appl. Genet. 1: 327; whichare incorporated herein by reference).

Selection for correctly targeted recombinants will generally employ atleast positive selection, wherein a nonhomologous expression cassetteencodes and expresses a functional protein (e.g., neo or gpt) thatconfers a selectable phenotype to targeted cells harboring theendogenously integrated expression cassette, so that, by addition of aselection agent (e.g., G418 or mycophenolic acid) such targeted cellshave a growth or survival advantage over cells which do not have anintegrated expression cassette.

It is preferable that selection for correctly targeted homologousrecombinants also employ negative selection, so that cells bearing onlynonhomologous integration of the transgene are selected against.Typically, such negative selection employs an expression cassetteencoding the herpes simplex virus thymidine kinase gene (HSV tk)positioned in the transgene so that it should integrate only bynonhomologous recombination. Such positioning generally is accomplishedby linking the HSV tk expression cassette (or other negative selectioncassette) distal to the recombinogenic homology regions so thatdouble-crossover replacement recombination of the homology regionstransfers the positive selection expression cassette to a chromosomallocation but does not transfer the HSV tk gene (or other negativeselection cassette) to a chromosomal location. A nucleoside analog,ganciclovir, which is preferentially toxic to cells expressing HSV tk,can be used as the negative selection agent, as it selects for cellswhich do not have an integrated HSV tk expression cassette. FIAU mayalso be used as a selective agent to select for cells lacking HSV tk.

In order to reduce the background of cells having incorrectly integratedtargeting construct sequences, a combination positive-negative selectionscheme is typically used (Mansour et al., Nature 336: 348-352 (1988)incorporated herein by reference). Positive-negative selection involvesthe use of two active selection cassettes: (1) a positive one (e.g., theneo gene), that can be stably expressed following either randomintegration or homologous targeting, and (2) a negative one (e.g., theHSV tk gene), that can only be stably expressed following randomintegration, and cannot be expressed after correctly targeteddouble-crossover homologous recombination. By combining both positiveand negative selection steps, host cells having the correctly targetedhomologous recombination between the transgene and the endogenous A-mybgene can be obtained.

Generally targeting constructs preferably include: (1) a positiveselection expression cassette flanked by two homology regions that aresubstantially identical to host cell endogenous A-myb gene sequences,and (2) a distal negative selection expression cassette. However,targeting constructs which include only a positive selection expressioncassette can also be used. Typically, a targeting construct will containa positive selection expression cassette which includes a neo genelinked downstream (i.e., towards the carboxy-terminus of the encodedpolypeptide in transnational reading frame orientation) of a promotersuch as the HSV tk promoter or the pgk promoter. More typically, thetargeting transgene will also contain a negative selection expressioncassette which includes an HSV tk gene linked downstream of a PGKpromoter.

FIG. 1 (bottom) is a schematic representation of a typicalpositive-negative A-myb targeting construct. FIG. 1 (bottom) shows theplacement of neo and tk (“TK”) genes. Arrows mark the transcriptionalorientation of the neo and tk genes. FIG. 1 (top) is a schematicrepresentation of the Hind III 5.9 kbp genomic clone used to generatethe targeting construct of FIG. 1 (bottom). Black boxes in therestriction maps of FIG. 1 represent exons.

The targeting construct of FIG. 1 (bottom) was deposited in the UnitesStates Department of Agriculture Northern Research Laboratories, Peoria,Ill. under accession number B21576 on May 1, 1996. The HindIII 5.9 kbpgenomic clone used to generate the targeting construct was deposited inthe same depository, on the same date, under accession number B21575.The nucleotide sequence of the 5.9 kbp clone is SEQ ID NO:3.

Typically, targeting polynucleotides of the invention have at least onehomology region that is at least about 50 nucleotides long, and it ispreferable that homology regions are at least about 75 to 100nucleotides long, and more preferably at least about 200-2000nucleotides long, although the degree of sequence homology between thehomology region and the targeted sequence and the base composition ofthe targeted sequence will determine the optimal and minimal homologyregion lengths (e., G-C rich sequences are typically morethermodynamically stable and will generally require shorter homologyregion length). Therefore, both homology region length and the degree ofsequence homology can only be determined with reference to a particularpredetermined sequence, but homology regions generally must be at leastabout 50 nucleotides long and must also substantially correspond or besubstantially complementary to a predetermined endogenous targetsequence. Preferably, a homology region is at least about 100nucleotides long and is identical to or complementary to a predeterminedtarget sequence in or flanking the A-myb gene. If it is desired thatcorrectly targeted homologous recombinants are generated at highefficiency, it is preferable that at least one homology region isisogeneic (i.e., has exact sequence identity with the crossover targetsequence(s) of the endogenous A-myb gene), and is more preferred thatisogeneic homology regions flank the exogenous targeting constructsequence that is to replace the targeted endogenous A-myb sequence.

The A-myb sequence may be scanned for possible disruption sites.Plasmids are engineered to contain an appropriately sized constructreplacement sequence with a deletion or insertion in the A-myb gene andat least one flanking homology region which substantially corresponds oris substantially complementary to an endogenous target DNA sequence.Typically, two flanking homology regions are used, one on each side ofthe replacement region sequence. For example, one homology region may besubstantially identical to a sequence upstream (i.e., the directiontowards the transcription start site(s) of the murine A-myb exon 4 and asecond homology region may be substantially identical to a sequencedownstream of the murine A-myb exon 4.

The A-myb gene is inactivated by homologous recombination in apluripotent cell line that is capable of differentiating into germ celltissue. A DNA construct, as discussed above, that contains an alteredcopy of a mouse A-myb gene is introduced into the nuclei of ES cells. Ina portion of the cells, the introduced DNA recombines with theendogenous copy of the mouse A-myb gene, replacing it with the alteredcopy. Cells containing the newly engineered genetic lesion are injectedinto a host mouse embryo, which is reimplanted into a recipient female.Some of these embryos develop into chimeric mice that possess germ cellsderived from the mutant cell line. Therefore, by breeding the chimericmice it is possible to obtain a new line of mice containing theintroduced genetic lesion.

Vectors containing a targeting construct are typically grown in E. coliand then isolated using standard molecular biology methods, or may besynthesized as oligonucleotides. Direct targeted inactivation which doesnot require prokaryotic or eukaryotic vectors may also be performed.Targeting constructs can be transferred to host cells by any suitabletechnique, including microinjection, electroporation, lipofection,biolistics, calcium phosphate precipitation, and viral-based vectors,among others. Other methods used to transform mammalian cells includethe use of Polybrene, protoplast fusion, and others (e.g., generally,Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., 1989,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which isincorporated herein by reference).

For making transgenic non-human animals (which include homologouslytargeted non-human animals), embryonal stem cells (ES cells) arepreferred. Murine ES cells, such as AB-1 line grown on mitoticallyinactive SNL76/7 cell feeder layers (McMahon and Bradley (1990) Cell 62:1073) essentially as described (Robertson, E. J. (1987) inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach. E. J.Robertson, ed. (Oxford: IRL Press), p. 71-112) may be used forhomologous gene targeting. Other suitable ES lines include but are notlimited to, the E14 line (Hooper et al. (1987) Nature 326: 292-295), theD3 line (Doetschman et al. (1985) J. Embryol. Exp. Morphi. 87: 27-45),and the CCE line (Robertson et al. (1986) Nature 323: 445-448). Thepractice of the present invention is specifically exemplifiedhereinafter using ES cells of mouse strain 129/J (Jackson Laboratories).The success of generating a mouse line from ES cells bearing a specifictargeted mutation depends on the pluripotence of the ES cells (i.e.,their ability, once injected into a host blastocyst, to participate inembryogenesis and contribute to the germ cells of the resulting animal).The blastocysts containing the injected ES cells are allowed to developin the uteri of pseudopregnant nonhuman females and are born as chimericmice. The resultant transgenic mice are chimeric for cells having aninactivated endogenous A-myb locus and are backcrossed and screened forthe presence of the correctly targeted transgene(s) by PCR or Southernblot analysis on tail biopsy DNA of offspring so as to identifytransgenic mice-heterozygous for the inactivated A-myb. By performingthe appropriate crosses, it is possible to produce a transgenic nonhumananimal homozygous for functionally disrupted A-myb alleles. Suchtransgenic animals are substantially incapable of making an endogenousA-myb gene product.

The functionally disrupted A-myb homozygous null mutant transgenicanimals will typically comprise rats or mice, but nonmurine species suchas dogs, cattle, sheep, goats, pigs and nonhuman primates, for example,may be utilized.

Homozygous null male A-myb animals are infertile due to a block inspermatogenesis. Histopathological examination of testes fromA-myb^(−/−) mice of the present invention indicates that thedifferentiation of spermatogonia is arrested at the pachytene stage ofmeiosis, indicating an essential role for A-myb in male germ celldifferentiation. It appears that A-myb is essential for transition ofspermatogonia to the spermatid stage. The results described herein showthat the proliferation of primary germ cells is not dependent on thepresence of A-myb but that their differentiation is dependent on thesynthesis of the A-Myb protein. Loss of A-Myb does not seem to affectthe formation of Leydig and Sertoli cells. Earlier studies (Mettus etal., Oncogene 9, 3077-3086, 1994) have shown that A-myb is not expressedin Sertoli cells or Leydig cells. These cells appear normal inA-myb^(−/−) mice.

The histopathology of the testis seen in A-myb^(−/−) mice issurprisingly similar to the histopathology seen in a large percentage ofmen who are infertile (Rosai, J., “Testis” in Ackerman's SurgicalPathology, G. Stamathis ed. (C. V. Mosby Co.), pp. 949-982, 1989;Soderstrom and Suominen, Arch. Pathol. Lab. Med. 104, 476-482, 1980;Wong, et al., Arch. Pathol. 95, 151-159, 1973). Biopsy specimens frominfertile men with total lack of spermatozoa usually show one of thefollowing conditions: (1) germ cell aplasia (Sertoli cell-onlysyndrome), in which the tubules are populated by Sertoli cells only andthere is a complete absence of germ cells; (2) spermatocystic arrest,characterized by a halt of the maturation sequence, usually at the stageof the primary and secondary spermatocyte development where nospermatids or spermatozoa are present; and (3) generalized fibrosiswhich appears to result in obstructive azoospermia due to bilateralobstruction or absence of some part of the duct system. The testicularhistopathology of A-myb^(−/−) mice is indistinguishable from thehistopathology of biopsies described in infertile men suffering fromspermatocystic arrest, suggesting that defects in A-myb expression orfunction may constitute the molecular basis of this form of humaninfertility.

The ovaries of the A-myb^(−/−) female mice appear normal by histologicalassessment which is further evidence by the ability of these mice tobecome pregnant and deliver pups. However, a striking abnormality wasobserved in the female A-myb^(−/−) mice following the birth of the pups.Examination of the mothers revealed complete absence of milk formationfollowing the delivery of pups. Pathological examination of theA-myb^(−/−) mothers 48 hours following delivery revealed severeimpairment of mammary epithelial proliferation in mutant mice followingpregnancy.

Development of mouse mammary epithelium occurs in two stages. The firststage of development occurs during puberty, when the breast tissuebecomes fully developed and is characterized by ductal elongation. Theductal cells at this stage express estrogen receptors and the ductalelongation is believed to be stimulated by estrogens. Following sexualmaturation, the mammary epithelial cells acquire progesterone receptorsand at this stage of development, require both estrogen and progesteronefor proliferation. During pregnancy, the combined action of estrogensand progesterone results in ductal side branching and lobuloalveolardevelopment. The mammary glands of A-myb^(−/−) mice appeared to developnormally during sexual maturation, as evidenced by histologicalanalysis. However, during pregnancy, the proliferation of mammaryepithelium was considerably diminished in the A-myb^(−/−) mice. Thisappeared to be due to reduced cell proliferation resulting in diminishedductile branching following pregnancy. These results indicate that A-mybmay play a critical role in steroid-induced proliferation of mammaryepithelium during pregnancy.

The A-myb^(−/−) male animals of the invention may be utilized as a modelfor male infertility, and for studying spermatogenesis. The A-myb^(−/−)animals may be used in the screening of potential therapeutic syntheticA-myb peptides. Such peptides could be screened for the ability toinduce resumption of spermatogenesis upon local administration to thetestes of A-myb^(−/−) mice. The candidate peptide would be administeredlocally by injection into the testes of the A-myb^(−/−) mice.

The A-myb^(−/−) animals of the invention may also be used as hostanimals for the transfer of desired transgenes via the sperm ofA-myb^(−/−) individuals rescued with a transgene construct encodingnon-defective A-myb gene and the desired additional transgene. TheA-myb^(−/−) animals described herein contain immature germ cells. Sincethese are stem cells, they have the potential for self renewal and thusundergo replication. These cells may be induced to replicate and undergodifferentiation to mature spermatids upon the introduction of exogenousA-myb.

Accordingly, spermatogonia are isolated from the A-myb^(−/−) animals,cultured in vitro and then transfected with a transgene constructcomprising a first DNA sequence encoding a functional A-Myb polypeptidewhich is linked to a second DNA sequence comprising the transgene ofinterest, the expression of which is desired in the animal's germline.The first and second DNA sequences may be operatively linked in that thetranscription of both sequences is under the control of the samepromoter or enhancer. Alternatively separate promoter/enhancer elementsmay be included in the construct for the first and second DNA sequences.

The transfected cells are then reintroduced into the testes of theA-myb^(−/−) animals. This will allow for expression of A-myb and thecontinuation of spermatogenesis, resulting in the production of viablespermatozoa which include the transgene of interest. While notnecessarily required, it is preferred that the spermatogonia donor andtransfected cell recipient are of the same species, preferably the samestrain.

The practice of the present invention is exemplified herein using theneo gene as the transgene. It may be appreciated that it is possible togenerate nonhuman animals producing sperm which harbor any desiredtransgene, provided the transgene may be contained in a constructfurther including a wild type A-myb gene. Spermatogonia which fail toincorporate the transgene construct encoding the wild type A-myb geneand the transgene of interest will not mature into spermatozoa.Spermatogonia which successfully incorporate the construct are “rescued”by the A-myb wild type transgene, but will also contain the additionaltransgene of interest. Since only immature germs cells which are“rescued” by the transgene construct are able to mature into spermatozoacompetent for fertilization, the sperm output of the rescued animal islimited to cells which have successfully incorporated the construct andthe transgene of interest. The A-myb^(−/−) animals “rescued” with thetransgene construct thus possess transgenic sperm, which can pass thedesired transgene to offspring. Alternatively, the transgenicspermatozoa can be harvested and used to artificially inseminatefemales, or to fertilize eggs in vitro.

Accordingly, spermatogonia are isolated from A-myb^(−/−) animals,cultured in vitro, and transfected with an appropriate “rescue”construct containing wild-type A-myb DNA and the transgene of interest.The transfected cells are then reintroduced into the testis ofA-myb^(−/−) animals.

Spermatogonia may be isolated from A-myb^(−/−) animals according toknown methods. Methods for isolating germ cells from the testis ofmammals are known to the art. See, e.g., Ogawa et al., J. Dev. Biol. 41,111-122 (1997); Brinster and Zimmermann, Proc. Natl. Acad. Sci. USA 91,11298-302 (1994); Brinster and Avarbock, Proc. Natl. Acad. Sci. USA 91,11303-7 (1994); Hofmann et al., Exp. Cell Res. 201:417-435 (1992);Hofmann et al., Proc. Natl. Acad. Sci. USA 91, 5333-7 (1994). The entiredisclosures of the preceding references are incorporated herein byreference.

The isolated spermatogonia are transfected with a rescue constructcontaining A-myb DNA encoding a functional A-Myb polypeptide, e.g. cDNA,under the control of a promoter to obtain A-myb expression, and atransgene of interest. The construct includes structural sequencesencoding the functional A-Myb polypeptide and the transgene of interest,and linked regulatory elements that drive expression of both structuralsequences in the host. At least one promoter/enhancer is linked upstreamof the first structural sequence in an orientation to drivetranscription of the A-myb wild-type DNA and transgene of interest. Thepromoter is selected so as to provide for expression of the transgenesin the testis. While the PGK2 and CMV promoter/enhancer elements areknown to be capable of driving constitutive gene expression in thetestis (Robinson et al., Proc. Natl. Acad. Sci. USA 86, 8437-41, 1989;Rosenberg et al., Cell Growth & Differ. 1995; Goto et al., Exp. CellRes. 186, 273-8, 1990), it is preferred that the promoter driveexpression at levels similar to the naturally occurring A-myb gene, andthe promoter is selected accordingly. Most advantageously, the promotercomprises the naturally occurring A-myb promoter.

The rescue construct generally encodes the full-length A-Myb protein, orfragment or analog thereof, having sufficient A-Myb activity to permitspermatogenesis to proceed in the host. Preferably, the A-Myb-encodingDNA encodes the full-length protein. The transgene of interest may bedesigned so as to provide for the expression of any desired gene in therescued host animal.

To express A-myb selectively rather than constitutionally, a 10 kbpHindIII genomic fragment containing the A-myb promoter region isisolated from a lambda phage mouse genomic library using ³²P-labeledA-myb cDNA as a probe. The HindIII genomic fragment is further cleavedwith BstEII to generate a HindIII/BstEII fragment containing the A-mybpromoter/enhancer region. The HindIII/BstEII fragment may be cloned intothe BstEII site of an A-myb cDNA clone.

The isolated A-myb^(−/−) spermatogonia may be transfected with therescue construct by methods known to those skilled in the art such as bythe calcium phosphate method (Chen and Okayama, Mol. Cell. Biol. 7,2745-52, 1987), the electroporation method (Potter, Anal. Biochem 74,361-373, 1988; Zheng and Chang, Biochim. Biophys. Acta 1088, 104-110,1991) the liposome-mediated method (Lopata et al., Nucl. Acids Res. 12,5705, 1984) or the DEAE-dextran method (Felgner et al., Proc. Natl.Acad. Sci. USA 84, 7413-7, 1987). The entire disclosures of theaforementioned references are incorporated herein by reference.

After overnight incubation at 37° C., the transfected cells are thenintroduced into the seminiferous tubules in the testes of A-myb^(−/−)mice, such as according to the procedure of Brinster and ZimmermannBrinster, supra. As an alternative to injection into the seminiferoustubules, the transfected cells may be injected into the rete testis(Ogawa et al., Int. J. Dev. Biol. 41, 111-122 (1997), the entiredisclosure of which is incorporated herein by reference). A thirdpossibility is to introduce the transfected cells by cannulating one ofthe five different ducts running from the rete testis to the head of theepididymis, thereby filling the rete and subsequently the tubules (Id.).

The mice are maintained for 2-6 months to allow the transfected stemcells to undergo spermatogenesis. Since A-myb^(−/−) lackspermatogenesis, the presence of mature spermatids and spermatozoa intransplanted mice would indicate successful integration of the rescueconstruct, and the transgene of interest. The rescued animals can thenbe mated to produce offspring containing the germ line transgene, or thetransgenic spermatozoa can be harvested and used to artificiallyinseminate females, or to fertilize eggs in vitro.

As an alternative to repopulation of A-myb^(−/−) mice with transfectedprimary spermatogonial cells, A-myb^(−/−) mice may be infected withretroviruses containing A-myb and the desired transgene. A retrovirusvector is constructed wherein A-myb cDNA is expressed under the controlof the A-myb promoter. The structure of one such vector designated pMV-7Δ ClaI/neo E/H-A-myb, wherein the transgene is the neo gene, is shown inFIG. 9C. Arrows in FIG. 9C indicate transcription start sites. It may beappreciated that the neo gene may be replaced in the vector of FIG. 9Cwith any transgene which one desires to incorporate into thespermatogonia cell DNA.

High titer viruses are then generated, such as according to the methoddescribed by Kozak and Kabat, J. Virol. 64, 3500-3508, 1990,incorporated herein by reference. It has been demonstrated thatrecombinant retroviruses of encoding c-myb cDNA may be generated intiters up to 10⁷ particles/ml (Patel et al., Mol. Cell. Biol. 13,2269-2276, 1993, incorporated by reference). Irradiated packaging cellsproducing virus, or concentrated preparation of virus, are injected intothe testes of anesthetized A-myb^(−/−) mice. Recipient mice aremaintained over a period of several months and analyzed for theproduction of mature sperm and spermatids. The rescued animals can thenbe mated to produce offspring containing the germ line transgene, or thetransgenic spermatozoa can be harvested and used to artificiallyinseminate females, or to fertilize eggs in vitro.

The present invention also provides treatment methods for restoringfertility in male individuals who are infertile due to a genetic defectin the A-myb locus. Such individuals are characterized by a low level ofA-Myb protein, or a loss of functional A-Myb protein. Such individualsmay be identified by an analysis of the A-Myb protein size, which willreveal mutations that block A-Myb synthesis, or gene rearrangementswhich result in production of a truncated protein. Deleterious pointmutations which result in a loss of the A-Myb protein's DNA bindingability can be identified by DNA binding assays using syntheticoligonucleotide binding sites as described by Golay et al., Oncogene 9,2469-2479 (1994), the entire disclosure of which is incorporated hereinby reference. Alternatively, A-myb cDNA from the afflicted individualmay be prepared and sequenced according to conventional techniques, andthe sequence compared to the wild-type human A-myb cDNA sequence (SEQ IDNO:5). The A-myb translation initiation codon in SEQ ID NO:5 comprisesnucleotides 105-107. The amino acid sequence of the human A-mybpolypeptide is set forth as SEQ ID NO:6. As the A-myb gene is expressedin peripheral blood cells, peripheral blood lymphocytes may beconveniently utilized as a source of A-myb DNA for analysis. Methods ofsequence analysis aimed at identifying mutations, e.g., the so-calledsingle-strand conformation polymorphism or “SSCP” method (Orita et al.,Genomics 5, 874-879, 1989, incorporated herein by reference) may beutilized to screen for A-myb mutations.

Local gene therapy is carried out to transfer to the cells of the testesof A-myb defective individuals a construct encoding a functional A-Mybpolypeptide. The transfer may be carried out by removing and engineeringspermatogonia of such A-myb^(−/−) individuals with a DNA constructdesigned to express a functional A-Myb polypeptide. The constructpreferably incorporates the complete coding segment of the human A-mybcDNA. The engineered spermatogonia are then returned to the testes ofthe infertile donor. Successful incorporation of the construct resultsin the production of functional A-Myb polypeptide in the testes of thesubject, and production of maturation of competent sperm.

Fertility may be restored in a subject who is infertile due to a defectin the A-myb locus through the use of a retrovirus vector directing theincorporation of DNA encoding a functional A-Myb protein intoappropriate cells of the testes. The retrovirus vector is used to infectthe testis of the individual in order to obtain the local production ofa functional A-Myb polypeptide.

While the functional A-Myb polypeptide which is expressed in the testesaccording to the aforesaid infertility treatment methods will typicallycomprise the full-length wild-type A-myb expression product, alsoincluded in the scope of the invention is the expression of polypeptideswhich comprise fragments of the complete naturally occurring geneproduct, or analogs thereof which differ from the latter by one or moreamino acid insertions, deletions and/or substitutions, provided suchfragments and analogs are functional in restoring fertility uponexpression in the host.

Infertility may be also be treated by the local administration to thetestes of an infertile A-myb^(−/−) individual an exogenous functionalA-Myb polypeptide. The polypeptide most advantageously comprises thefull-length wild-type A-myb expression product, but the functional A-Mybpolypeptide may comprise an A-Myb fragment or analog, as describedabove. Such vehicles may comprise, for example, aqueous vehicles such asnormal saline.

The dosage and treatment schedule are selected so as to provide forcontinuous production of viable sperm at a level which can supportsuccessful fertilization.

Also included in the scope of “functional A-Myb” polypeptide is a fusionproduct comprising the naturally occurring A-Myb polypeptide or analogthereof and one or more attached amino acid sequences which enhance thecellular uptake or penetration of the A-Myb polypeptide into the cellsof the testes. Such fusion products may be prepared with resort tocommercially available expression vectors which provide forincorporation of DNA sequences of interest downstream from a DNA segmentencoding an amino acid sequence having desirable transport properties.The resulting A-Myb fusion protein may be used as the exogenouslysourced functional A-Myb protein in treating A-myb^(−/−) individuals forinfertility.

Alternatively, a cell-penetrating peptide may be ligated directly to theN-terminal or C-terminal portion of the functional A-Myb polypeptide toenhance uptake. One such peptide is the sixteen amino acid peptide fromthe third helix of the Antennapedia homeodomain,Arg-Gln-Ile-Lys-Ile-Phe-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys (SEQ IDNO:7) (Derossi et al., J. Biol. Chem. 269 (14):10444-10450, 1994). This16 amino acid peptide, commercially available as “Penetratin 1™” fromAppligene, Inc., may be coupled to a functional A-Myb polypeptideaccording to the manufacturer's protocols (Penetratin 1™, Appligene,Inc., 1177C Quarry Lane, Pleasanton, Calif. 94566).

The practice of the invention is illustrated by the followingnonlimiting examples. As it relates to the rescue of nonhumanA-myb^(−/−) animals from infertility by incorporation of a constructencoding wild-type A-myb DNA and a transgene of interest, the practiceof the present invention is exemplified using the neo gene, a bacterialgene, as the transgene. The same neo gene also provides for positiveselection of transformants harboring an integrated “rescue” constructencoding. It should be understood that neo DNA may be replaced by anyother transgene, or that additional transgenes of interest may beincluded in addition to neo such that the positive selection function ofthe neo gene product may be retained in screening transformants.

EXAMPLE 1 Preparation of A-myb Knockout Mice

A. Isolation of a Murine A-myb Genomic Clone

The mouse A-myb gene was isolated as follows by screening a λ DASH mousegenomic library derived from the 129/J mouse strain, using a probederived from the 5′ end of the A-myb cDNA clone that encodes the DNAbinding domain of the A-Myb protein.

Accordingly, DNA obtained from the tissue of a 129/J mouse (JacksonLaboratories) was digested with the following restriction enzymes:BamHI, EcoRI, HindIII, PstI and XbaI. From a Southern analysis of thedigests, the DNA was digested with HindIII to obtain a genomic clone. Anapproximately 800 bp A-myb cDNA probe was prepared consisting of anEcoRI fragment from the 5′ end of the A-myb cDNA spanning cDNAnucleotide positions 1-794 of the A-myb mouse cDNA. The mouse A-myb cDNAnucleotide sequence and deduced amino acid sequence of the A-Myb proteinare shown in SEQ ID NO:1 and 2, respectively. The digested genomicSouthern blot was probed with the A-myb cDNA probe. A 5.9 kbp Hind IIIfragment was cloned by size selection in a sucrose gradient followed bycloning into Hind III digested Lambda DASH II phage (Stratagene,LaJolla, Calif.). From the resulting library, the 5.9 kbp A-myb fragmentwas cloned and subcloned into the pGEM 7Zf(+) plasmid vector (PromegaCorp., Madison, Wis.) according to standard procedures (MolecularCloning: A Laboratory Manual, T. Maniatis, E. Fritsch and J. Sambrook,eds. (1982), Cold Spring Harbor Laboratory). The structure of thegenomic clone and corresponding restriction map of mouse A-myb is shownin FIG. 1. Analogous to c-myb, A-myb contains three tandem amino aciddirect repeats which make up a DNA binding domain. The genomic clone wascompletely sequenced (SEQ ID NO:3) according to the method of Sanger etal., Proc. Natl. Acad. Sci. USA 74, 5463-5467 (1977) and found tocontain A-myb exons 3, 4 and 5 (FIG. 1, black boxes). Exon 3 encodes the5′ end of the first repeat of the DNA binding domain, while exon 4encodes the 3′ end of the first repeat and the 5′ end of the secondrepeat. Exon 5 encodes the 3′ end of the second repeat and the 5′ end ofthe third repeat. The genomic clone was deposited as NRRL B-21575 on May1, 1996.

B. Preparation of A-myb Knockout Targeting Vector

To produce a null allele of A-myb, a gene targeting vector was preparedfrom the 5.9 kbp genomic clone by following the positive-negativeselection method which was originally described by Thomas and Capecchi,Cell 51:503-512 (1987), the entire disclosure of which is incorporatedherein by reference. The vector was designed to disrupt the DNA bindingdomain of the A-myb gene by insertion of the neomycin transferase (neo)gene into A-myb exon 4 at the ClaI site (FIG. 1).

Accordingly, the Pgk-Neo gene (Mansour et al., Nature 336, 348-352(1988), incorporated herein by reference) was digested with EcoRI andHindIII to yield a 2.0 kbp neo cassette. This DNA fragment was filled byusing the Klenow fragment of DNA polymerase I. The genomic clone ofA-myb was digested with ClaI, blunt ended and then ligated with the DNAfragment containing the neo cassette. Insertion of the neo cassetteresults in the disruption of the gene which codes for the DNA-bindingdomain of the A-Myb protein. The EcoRV/HindIII fragment of this clonewas then released by digestion with HindIII and EcoRV, filled using theKlenow fragment of DNA polymerase I and blunt end ligated to the Pgk-TKvector (Thomas and Capecchi, supra) at the HindIII site. The resultingtarget vector is shown in FIG. 1 (bottom). The orientation of the A-myb,neo and tk genes was determined by restriction endonuclease analysis inconjunction with DNA sequence analysis. The orientation of the neo andtk genes is indicated by arrows in FIG. 1. The neo cassette containedthe neomycin transferase gene under the control of the phosphoglycerolkinase 1a promoter and polyadenylation signals. The thymidine kinasegene was flanked by the same promoter and polyadenylation signals.

C. Incorporation of Target Vector in ES Cells

Mouse ES cells (cell line E14a, Handyside et al., Roux's Arch. Dev.Biol. 198, 48-55, 1989) were maintained as previously described byRobertson, “Embryo-derived Stem Cell Lines” in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed.(Oxford, 1987; IRL Press), pp. 71-112, and cultured in Dulbecco'sModified Eagle's Medium supplemented with 15% fetal calf serum (SigmaChemical Co., St. Louis, Mo.) and 1000 U/ml of leukemia inhibitoryfactor on γ-irradiated SNL feeder layers. The above-prepared A-mybknockout targeting vector was linearized with PvuI and introduced intothe mouse ES cells by electroporation (250V, 500 mF) as described byThomas and Capecchi, supra. Following electroporation, the ES cells wereplated into 6 cm plates containing G418 (0.1 mg/ml. active ingredient)and 2 μM ganciclovir. On days 9 and 10 of selection, individual cloneswere picked, dispersed into single cell suspension in 0.25% trypsin andseeded into two wells, each in a separate 48 well tissue culture plate.After 5 to 7 days, one plate was used for DNA isolation for Southernblot analysis by standard protocols.

DNA was isolated from 87 double resistant clones, digested with HindIII,blotted onto nitrocellulose membranes, and probed with a ³²P-labeled 700bp SspI 5′ A-myb probe. The probe hybridizes to a 5.9 kbp fragmentderived from the wild-type A-myb allele, while the targeted locus ispredicted to yield an 8 kbp fragment. The results are shown in FIG. 2.The positions of the molecular weight markers (γDNA digested withHindIII) is shown on the left. Lanes 4 and 5 contain the DNA from EScell clones that contain a disrupted A-myb locus. The appearance of an 8kbp band indicates a homologous recombination event in two of the 87clones analyzed. Thus, the mouse A-myb gene was targeted using 4.5 kb ofhomologous sequence using isogeneic DNA at a frequency of approximately1 in 40 of the doubly selected clones analyzed.

D. Production of Mouse Chimeras With Heterozygous Null A-myb ES Cells

The two clones bearing a disrupted A-myb gene were microinjected intoC57/B6 blastocysts (10-15 cells/embryo), transferred to pseudopregnantfoster CD1 females and male chimeras were produced. The chimerasappeared normal and were mated with females from C57/B6 to obtainheterozygous (A-myb^(+/−)) mice. The mice appeared normal. Eightoffspring with agouti color were produced. Southern blot analysis of DNAderived from tail biopsies of these mice showed that three of them wereheterozygous for the A-myb disruption.

E. Production of Homozygous A-myb^(−/−) Null Mice

Intercrosses were set up between mice heterozygous for the disruptedA-myb allele and progeny were analyzed for A-myb genotype ten days afterbirth by Southern blot analysis of DNA derived from tail biopsies. TheDNA was extracted from the tail biopsies of the 10-day old pups,digested with HindIII, and blotted onto a nitrocellulose membrane. Theblots were hybridized with the ³²P-labeled 700 bp SspI probe. Genotypingwas determined according to the sizes of the Hind III fragmentshybridized (FIG. 3): A-myb^((+/+)), 6 kb; A-myb^(+/−), 6 kb and 8 kb;A-myb^(−/−), 8 kb. The progeny generated were in the expected Mendellianratio of 1:2:1 (103:237:91) for wild type (+/+), heterozygous (+/−), andhomozygous (−/−) A-myb alleles.

F. Homozygous A-myb^(−/−) Mouse Phenotype

(i) Dwarfism

All A-myb^(−/−) pups showed a similar appearance. At birth, A-myb^(−/−)mice appeared to be indistinguishable from their littermates. Adifference in size and appearance of A-myb^(−/−) mice was seen duringthe first few weeks of life. The A-myb^(−/−) pups were runted, wrinkled,and exhibited hunched posture as compared to their littermates. Such anappearance was not observed with the A-myb^(+/+) and A-myb^(+/−) pups.No difference was observed between the heterozygous and wild type A-mybpups. At four weeks, the A-myb^(−/−) pups were approximately 40% thesize of their A-myb^(+/−) littermates. As the A-myb^(−/−) mice matured(up to three months), their runted and hunched posture appearance becameless pronounced and the mice almost attained the body size (as measuredby total body weight) that is comparable to the A-myb^(+/+) andA-myb^(+/−) mice (almost 90% for the females and approximately 70% forthe males). However, 25% of the mice do not survive after three to fourweeks after birth (23 out of 91).

(ii) Behavioral Defects.

All of the A-myb^(−/−) pups initially displayed the same pattern ofaltered behavior. These pups exhibit behavioral alterations such asincreased hyperactivity (as exemplified by frantic running), tremblingupon suspension by the tail, bat and ball postures with hindlimbcrossing upon suspension by tail, hunched posture, and inclination tobite handler. Five-six week old males exhibited increased aggressivebehavior when mated with female mice for the first time. Females matedwith the A-myb^(−/−) male mice had noticeable bite wounds on thegenitals, rump, and tail within days after matings were set up. However,older (10-12 weeks old) A-myb^(−/−) male mice do not exhibit thisparticular behavior during matings even after a period of several weeks.The A-myb^(−/−) female mice have not shown such enhanced aggressivebehavior even though they remained hyperactive. All the above abnormalbehavioral patterns exhibited by the A-myb^(−/−) mice lessened inseverity as the mice matured to 3 months of age. Pathologicalexamination of the nervous system did not reveal any obviousabnormalities in the brain, spinal cord or other structures. However,the possibility of minor neuroanatomical defects that could lead to someof these neurological abnormalities can not be ruled out. While theA-myb^(−/−) female mice are fertile, the A-myb^(−/−) male mice areinfertile but are able to copulate as evidenced by the formation ofvaginal plugs in the females after mating.

(iii) Body and Testes Size

FIG. 5A is a comparison of the total body size and testicular size of 4week-old A-myb^(+/−) (left) and A-myb^(−/−) (right) mice from the samelitter. The testes dissected from the same mice are shown below themice. FIG. 5B is a comparison of the total body size and testicular sizeof 10 week-old A-myb^(+/+) (left), A-myb^(+/−) (middle) and A-myb^(−/−)(right) mice from the same litter. The testes dissected from the samemice are shown below the mice. The testis of A-myb^(−/−) are atrophic.The size and weight of the testis from the A-myb^(−/−) mice isapproximately 25% of their littermates. The small size of the testis isnot due to the fact that these A-myb^(−/−) mice are, at least in theinitial weeks after birth, smaller in body size as compared to theirheterozygous and wild type A-myb littermates (FIG. 5A). When the ratioof testis weight versus total body weight was compared, it became clearthat there was a four-fold reduction in the weights of testes derivedfrom the A-myb^(−/−) mice as compared to their heterozygous and wildtype litter mates. Even after the male mice attained a more normalweight as a function of age, the testes failed to recover weight unlikeother organs and remained in an atrophic state (FIG. 5B).

(iv) Sperm Count

Sperm counts, using whole testis preparations (Amann et al., Biol.Reprod. 15, 586-592, 1976), showed that there was a complete absence ofspermatozoa in A-myb^(−/−) testes, whereas A-myb^(+/+) mice contained23.66±1.1×10⁶ spermatozoa per testis, and A-myb^(+/−) mice contained22.8+1.26×10⁶ spermatozoa per testis.

(v) Lack of Spermatogenesis

The following pathological analysis of the atrophic testis of theA-myb^(−/−) mice showed a lack of active ongoing spermatogenesis inthese mice. Tissues from mutant (A-myb^(−/−)), heterozygous(A-myb^(+/−)) or wild type (A-myb^(+/+)) mice were fixed in 10% bufferedformalin overnight, processed and embedded in paraffin using standardprocedures. Sections (4-8 μM) were cut and stained with hematoxylin andeosin according to standard procedures. FIG. 6A shows the hematoxylinand eosin-stained sections of the seminiferous tubules of A-myb^(+/+)mouse testes while FIG. 6B shows similar sections from A-myb^(−/−) mousetestis. Magnification is 100 ×.

In the A-myb^(+/+) mice (FIG. 6A), the differentiation of spermatozoaproceeds in a step-wise manner where the spermatogonia are located closeto the basement membrane, while cells at progressively later stages ofdifferentiation are situated closer to the tubular lumen. In the centerof the tubular lumen mature spermatozoa with tails can be distinguishedfrom other cell types. A-myb^(+/−) mice showed the identical pattern asthat of A-myb^(+/+) mice (data not shown).

A-myb^(−/−) mice lack spermatids and mature spermatozoa in the center ofthe lumen (FIG. 6B). The tubules contain primary and secondaryspermatogonia near the basement membrane. A small number of cells withsmall nuclei, with condensed and possible apoptotic nuclear chromatinare seen in some tubules which represent abortive secondaryspermatocytes. The differentiation of spermatogonia seemed to come to anabrupt halt at the pachytene stage of meiosis. Loss of A-myb did notseem to affect the formation of Leydig and Sertoli cell populations asthey appeared to be normal in A-myb^(−/−) mice. Thus, the appearance ofthe small sized testes in A-myb^(−/−) mice is accompanied by the arrestof spermatogenesis resulting in a total absence of mature spermatids andspermatozoa. These results suggest that while A-Myb synthesis is notessential for development primary and secondary spermatogonia, itsabsence results in a block to spermatogenesis immediately prior tomeiosis, at the pachytene stage. When the A-myb^(−/−) male mice wereallowed to mate with female mice, they were found to exhibit a normalsexual libido and copulated normally as evidenced by the formation ofvaginal plugs in the female mice. But as expected, none of the femalemice became pregnant. These results show that the infertility isassociated with the absence of spermatogenesis and not an absence ofsex-drive. The ovaries of the A-myb^(−/−) female mice appeared normalfrom histological staining, and as evidenced by the ability of thesemice to become pregnant.

(vi) Lack of A-Myb Protein Production in Male Mice

To verify that the A-myb protein was not synthesized in A-myb^(−/−)mice, Western blot analyses were performed. Since A-myb is predominantlyexpressed in the testis, homogenates were prepared using testesdissected from A-myb^(+/+), A-myb^(+/−) and A-myb^(−/−) mice.Accordingly, tissue samples were homogenized by Dounce disruption inlysis buffer (10 mM HEPES, pH 7.9; 1 mM EDTA; 60 mM KCl; 0.5% NP40; 1 mMDTT; 1 mM PMSF; 0.5 μg/ml of Leupeptin; 0.5 μg/ml of Pepstatin A and 0.5μg/ml of Aprotinin), clarified by centrifugation and the proteinconcentration in the supernatants was determined. Protein samples wereseparated by SDS-PAGE (10% polyacrylamide) and transferred tonitrocellulose membranes. Membranes were blocked by incubation in PBScontaining 2% non-fat dry milk, 2% BSA and 0.1% Tween 20 for 1 hour atroom temperature and then rinsed several times in T-TBS (0,05% Tween 20,20 mM Tris-HCl, pH 7.5, and 150 mM NaCl). The membranes were thenincubated with a 1:100 dilution of rabbit polyclonal antibody raisedagainst the DNA binding domain of the Myb protein for 1 hour at roomtemperature and then rinsed several times in T-TBS. The membranes werethen incubated with a 1:1000 dilution of the developing antibody (goatanti-rabbit Ig) for 30 minutes at room temperature and then rinsedseveral times in T-TBS. The membranes were then developed according tothe supplier by chemiluminescence (ECL). A band corresponding to theA-Myb protein in the A-myb^(+/+) and A-myb^(+/−) mouse testis lysateswas detected (FIG. 4). The band was totally absent in the A-myb^(−/−)testis lysate (FIG. 4).

(vii) Defects in the Proliferation of the Mammary Epithelium DuringPregnancy of A-Myb^(−/−) Female Mice

The ovaries of the A-myb^(−/−) female mice appeared histologicallynormal. FIG. 7A shows a cross-section of wild-type mouse ovaries whileFIG. 7B shows the cross-section of A-myb^(−/−) mouse ovaries.Magnification is 25×. Both cross-sections show normal primary andsecondary follicles with normal oocytes. The ovaries of the A-myb^(−/−)mice are proportionally smaller than their A-myb^(+/+) or A-myb^(+/−)littermates.

When the A-myb^(−/−) female mice were allowed to mate with wild-type orA-myb^(+/−) mice, they became pregnant and produced litters of normalnumber. Two days following delivery of pups, maternal female mice weresacrificed and then breast tissue examined for the development ofsecondary (pregnancy induced) alveolar and lobular growth anddevelopment. A dramatic abnormality in mammary function was apparent.A-myb^(−/−) female mice were found to be unable to nurse the pupsbecause of defective mammary tissue proliferation. The mammaryepithelium of wild type and A-myb^(+/−) mice underwent a massiveexpansion following pregnancy (FIG. 8A, 8B), which was considerablydiminished in A-myb^(−/−) mice (FIG. 8C, 8D). Specifically, the amountof breast tissue was markedly decreased when compared to a lactatingheterozygous mouse. This appeared to be due to diminished secondary(pregnancy induced) alveolar and lobular growth and development (FIG.8C, 8D). An intermediate lobular pattern was observed, which was closerto a resting lobular pattern. Mammary epithelial ducts from null mutantmice failed to significantly branch and develop alveolar structureswhich normally cluster together to form lobules that fill the mammaryfat pad (FIG. 8C, 8D).

EXAMPLE 2 Preparation of Fertile Transgenic Mice from A-myb KnockoutMice Rescued by Repopulation with Primary Spermatogonial CellsTransfected with A-myb and Transgene

A. Isolation of A-myb^(−/−) Primary Spermatogonia

Germ cells are isolated from the testis of A-myb^(−/−) mice as follows.Testes from male A-myb^(−/−) mice are removed at postnatal day 10. Thetunica are removed from the testes and the exposed tubules are treatedwith collagenase (1 mg/ml 15 min at 37° C.) followed by trypsindigestion (0.25% for 10 min. at 37° C.). The cells are then centrifugedat 600×g at 16° C. for 5 min. A cell fraction enriched with preleptotenespermatocytes is then isolated by the STA-PUT procedure and unit gravitysedimentation at 4° C. as described by Brinster and Zimmermann, Proc.Natl. Acad. Sci. USA 91, 11298-302 (1994). The cells are thenresuspended in CMRL-1066 medium supplemented with 80 μg/ml insulin, 3μg/ml transferrin, 80 μg/ml ascorbic acid and 13% fetal bovine serum.

B. Preparation of A-myb Retroviral Rescue Vector

A rescue construct, encoding a functional A-myb polypeptide and neo as atransgene, is prepared as follows.

1. Preparation of 5′ upstream A-myb DNA segment. A 10 kbp HindIIIgenomic murine A-myb fragment is isolated from a lambda phage mousegenomic library using ³²P-labeled A-myb cDNA as a probe. The 10 kbpHindIII fragment is further cleaved with BstEII to generate aHindIII/BstEII fragment (SEQ ID NO:4, nucleotides 1-3383) which containsthe A-myb promoter/enhancer region. A murine cDNA clone containing theA-myb 5′-untranslated region (designated clone 100; Mettus et al.,Oncogene 91, 3077-3086, 1994)) is isolated from a murine cDNA libraryusing as a probe a 1273 bp HpaII-NcoI fragment derived from the5′-region of the human A-myb cDNA (Nomura et al., Nucleic Acids Res. 16,11075-11089, 1988). Clone 100 is digested with BstEII and ClaI to form a0.3 kbp BstEII/ClaI cDNA fragment containing the A-myb 5′ -untranslatedregion (SEQ ID NO:4, nucleotides 3384-3730). The 3.4 kbp HindIII/BstEIIgenomic fragment containing the A-myb promoter is ligated to the 0.3 kpbBstEII/ClaI cDNA fragment to form a 3.7 kpb HindIII/ClaI fragment (SEQID NO:4, nucleotides 1-3730).

2. Preparation of A-myb DNA Coding Segment

A clone, designated pcDNA3_PGC2_A-myb, containing the A-myb codingsequence and a polyadenylation signal from the bovine growth hormonegene, is prepared as follows:

a. Preparation of pcDNA3. Vector pcDNA3.1v (Invitrogen Corp., 3985 BSorrento Valley Blvd., San Diego, Calif. 92121) is digested with NruIand HindIII to remove the CMV promoter. The ends of the resulting 4.7 kbfragment are filled by T4 polymerase treatment.

b. Preparation of PGK2. A 1.4 kb HindIII fragment is isolated from aPGK2/chloroamphenicol acetyl transferase (CAT) fusion gene, thepreparation of which is described by Robinson et al., Proc. Natl. Acad.Sci. USA 86(21):8437-41, 1989, the entire disclosure of which isincorporated herein by reference. The 1.4 kb HindIII fragment is bluntended by T4 polymerase treatment. The fragment contains the human PGK2promoter.

c. The PGK2 fragment of (b) is ligated into the pcDNA3 fragment of (a)by blunt end ligation. This results in the construct pcDNA3_PGK2.

d. Preparation of A-myb fragment. A construct containing full lengthA-myb coding sequence in the vector pGEM-37f (Promega Corp., 2800 WoodsHollow Rd., Madison, Wis. 53711-5399) is digested with HindIII andBstEII followed by PvuI and treatment with T4 polymerase (to blunt endthe fragment). The 2.6 kb A-myb coding sequence (HindIII/BstEII) ispurified.

e. The 2.6 kb A-myb coding sequence of (d) is cloned into theEcoRV-digested pcDNA3_PGK2 construct.

f. The proper orientation of all portions of the final construct,pcDNA3_PGK2 A-myb is verified by sequence analysis.

g. The PGK2-A-myb-bovine polyadenylation fragment(from the pcDNA3sequence) is removed by AatII/BsaAI digestion and isolation of the 5.1kb band.

h. The 5.1 kb band is then digested with ClaI and BsaAI to form a 3.0kbp ClaI/BsaAI fragment comprising SEQ ID NO:4, nucleotides 3371-6775.The A-myb coding sequence constitutes SEQ ID NO:4 nucleotides 3506-6134.The bovine growth hormone polyA signal from the pcDNA3 vectorconstitutes SEQ ID NO:4 nucleotides 6135-6775.

3. Preparation of Modified pMV-7 Retroviral Vector

The pMV-7 retroviral vector (Kirschmeier et al., DNA 7:219-225, 1988,incorporated herein by reference) contains the selectable drugresistance gene neo under the regulation of the herpes simplex virus(HSV) thymidine kinase (tk) promoter and unique EcoRI and HindIIIcloning sites for the insertion of cDNAs whose transcription isregulated by the 5′ long terminal repeat (LTR). See FIG. 9A for adiagram of pMV-7. To generate a rescue vector in which the A-myb cDNA isregulated by the A-myb promoter, the neo cassette is excised from pMV-7with ClaI digestion. This neo cassette is then placed in theEcoRI/HindIII cloning site in the pMV-7 vector to form the vector pMV-7Δ ClaI/neo E/H (FIG. 9B). This vector contains a ClaI site away from theLTR in which the A-myb cDNA may be introduced under the regulation ofthe A-myb promoter. The 3.7 kbp HindIII/ClaI fragment containing theA-myb promoter and 5′-untranslated region is ligated to the 3.0 kbpClaI/BsaAI fragment of the pcDNA3_PGK2_A-myb clone containing the A-mybcoding sequence and a polyadenylation signal form the bovine growthhormone gene. The resultant 6.7 kbp fragment (SEQ ID NO:4, nucleotides1-6775) is blunt ended and cloned into the ClaI site (also blunt ended)of the modified pMV-7 retroviral vector, pMV-7 Δ ClaI/neo E/H. Theorganization of the resultant fully-constructed A-myb retroviral rescuevector, designated pMV-7 Δ ClaI/neo E/H-A-myb, is shown in FIG. 9C.

C. Transfection of A-myb^(−/−) Primary Spermatogonia with RescueConstruct

Primary spermatogonia are obtained from testes of A-myb^(−/−) mice from5 to 60 days after birth by using a two-step enzymatic digestionprotocol as described by Ogawa et al., Int. J. Dev. Biol. 41:111-122(1997). The seminiferous tubules are first exposed by peeling the tunicaalbuginea from the testes. To disperse the tubules the testes are thenincubated in 10 volumes of Hanks' balanced salt solution without calciumor magnesium (HBSS) containing 1 mg/ml collagenase at 37° C. with mildagitation for 15 minutes. The tubules are then washed 4 times in 10volumes of HBSS, followed by incubation at 37° C. for 5 minutes in HBSScontaining 1 mM EDTA, 200 to 700 μg/ml DNAse and 0.25% trypsin with mildagitation. Trypsin activity is then terminated by the addition of 20%volume of fetal bovine serum. Large pieces of undigested material areremoved and the cell suspension is filtered through a nylon mesh with 60μm pore size to remove large clumps of cells. The filtrate is thencentrifuged at 600×g for 5 minutes at 16° C. The supernatant is thencarefully removed from the cell pellet. The cells are then resuspendedin Dulbecco's modified medium containing 10% fetal bovine serum. Thecells are then transfected with the A-myb rescue retroviral vector pMV-7Δ Cla/neo E/H-A-myb. The primary spermatogonia may be transfected byeither electroporation (Potter, Anal. Biochem. 74:361-373, 1988; Zhengand Chang, Biochim. Biophys. Acta 1088:104-110, 1991) or by liposomemediated transfection (Felgner et al., Proc. Natl. Acad. Sci U.S.A.84:7413-7414, 1987), according to the following protocols:

1. Transfection by electroporation:

5×10⁶ primary spermatogonia are washed with ice cold 1×phosphatebuffered saline (PBS) and then centrifuged for 5 minutes at 600×g at 4°C. The cell pellet is then resuspended in PBS at 1×10⁷ cells/ml. 0.5 mlof the cell suspension is then transferred into an electroporationcuvette at 0° C. The A-myb rescue retroviral vector DNA (1 to 10 μg) isadded, mixed and incubated on ice for 5 minutes. The cuvette is thenplaced in the electroporation apparatus at room temperature and isshocked at 1.5 kV at 25 μF. The cuvette is then incubated on ice for 10minutes. The transfected cells are then diluted 20-fold in Dulbecco'smodified medium containing 10% fetal bovine serum and incubated for 48hours at 37° C. in a CO₂ incubator. Stable transfected primaryspermatogonia expressing A-myb are then selected for by the addition ofG418 (400 μg/ml) to the medium.

2. Liposome-mediated transfection:

5×10⁵ primary spermatogonia are placed in 6 well dishes and placed at37° C. in a CO₂ incubator overnight. The A-myb rescue retroviral vectorDNA (1 to 10 μg) is complexed with the liposome in a polystyrene tube.The DNA is first diluted into 1 ml Dulbecco's modified medium, vortexedfor 1 second, and then the liposome suspension (10 μl) TransfectACE(GIBCO/BRL) and vortexed again for 1 second. The suspension is thenincubated at room temperature for 10 minutes. 1 ml of the DNA/liposomecomplex is then added directly to the cells and incubated at 37° C. in aCO₂ incubator for 48 hours. Stable transfected primary spermatogoniaexpressing A-myb are then selected for by the addition of G418 (400μg/ml) to the medium.

D. Transplantation of Transfected Cells

The transfected cells are transferred into the recipient A-myb^(−/−)mice, according to the protocol as described by Brinster and Zimmermann,supra. The mice are first anesthetized, and under a dissectingmicroscope, the testes are exposed and immobilized to align theseminiferous tubules with an injection glass pipette (1-mm outsidediameter and a 40 μm tip). Small incisions (1-3 mm) are made in thetunica to expose the seminiferous tubules. The tubules are entered withthe tip of the pipette and the cell suspension is injected into thetubule. The mice are maintained for 2 to 6 months to allow thetransfected stem cells to undergo spermatogenesis. Since A-myb^(−/−)mice lack spermatogenesis, the presence of mature spermatids andspermatozoa indicates that the A-myb transfected into the primaryspermatogonial cells is able to reinitiate spermatogenesis in thesemice.

EXAMPLE 3 Preparation of Fertile Transgenic Mice from A-myb KnockoutMice Rescued with Retroviruses Containing A-myb and Transgene

A vector is constructed by removing the CMV promoter from the vectorpMV7 by BamHI/HpaI and inserting the A-myb cDNA with the A-myb promoterat the same site (FIG. 9C). The vector contains the neo gene as thetransgene. High titer viruses using the “Ping-Pong amplification method”described by Kozak and Kabat, J. Virol 64, 3500-3508 (1990) aregenerated. Briefly, DNA constructs encoding A-myb are first transfectedinto Ψ-2 ecotropic packaging cells using calcium phosphateprecipitation. Forty-eight hours after transfection, the medium is usedto infect PA317 amphotropic packaging cells Miller and Rosman,Biotechniques 7, 980-90 (1989), incorporated herein by reference. Thismethod is repeated for 3-4 rounds and at the end of last infection,single cell clones of the virus producing cells are selected using G418resistance as a parameter of virus titer. Culture supernatants with highvirus titer are used to infect the testes of A-myb^(−/−) mice.Accordingly, recipient A-myb^(−/−) mice are anesthetized and the testisare injected with G418 resistant cells previously irradiated andcontaining A-myb expression vector or concentrated preparations of thevirus. The mice are maintained for over a period of several months andanalyzed for the production of mature sperm and spermatids by (a) theirability to impregnate female mice or (b) by histochemical analyses.

All references cited with respect to synthetic, preparative andanalytical procedures are incorporated herein by reference.

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof and,accordingly, reference should be made to the appended claims, ratherthan to the foregoing specification, as indication the scope of theinvention.

What is claimed is:
 1. A transgenic mouse having a genome comprising ahomozygous, functionally disrupted A-myb gene, wherein the mouse is aninfertile male.
 2. A mouse embryonic stem cell having a genomecomprising a homozygous, functionally disrupted A-myb gene, wherein saidembryonic stem cell is capable of becoming the transgenic mouse ofclaim
 1. 3. A method for generating the mouse embryonic stem cell ofclaim 2, comprising: transferring a targeting construct into embryonicstem cell; and selecting for the mouse embryonic stem cell according toclaim 2 having the targeting construct integrated into the endogenousA-myb gene of the mouse embryonic stem cell.
 4. The mouse embryonic stemcell according to claim 2, wherein said disrupted A-myb gene isdisrupted by an integrated targeting construct.
 5. The mouse embryonicstem cell according to claim 4, wherein the integrated targetingconstruct comprises a neo gene.
 6. The transgenic mouse according toclaim 1, wherein said disrupted A-myb gene is disrupted by an integratedtargeting construct.
 7. The transgenic mouse according to claim 6,wherein the integrated targeting construct comprises a neo gene.
 8. Thetransgenic mouse according to claim 1 which does not produce A-mybprotein.